Project Summary Bacterial pathogens have developed remarkable strategies to usurp host cell processes to establish infection, evade immunity, and disseminate to distal sites in the host. A key highly evolved virulence strategy shared by major human bacterial pathogens is the conscription of components of the host hemostasis system to assist survival of the microorganism against host innate defenses. One important human pathogen, Gram+ Group A Streptococcus pyogenes (GAS), is a model for employing such a tactic. There are >250 surface-expressed M- protein (M-Prt)-based serotypes of GAS that colonize pharyngeal and skin epithelial cells, and which possess the full range of minor-to-severe virulence. Many of these GAS strains contain highly expressed M-Prts, viz., PAM, that directly interact with host human plasminogen (hPg), whereas the M-Prts of some other strains (e.g., M1, M3) first interact with host fibrinogen (hFg), thus enabling the subsequent binding of hPg to the cells. These GAS cells also secrete coevolved subforms of streptokinase (SK) that specifically activate GAS-bound hPg to the serine protease, plasmin (hPm). In this manner, an active plasma inhibitor-resistant protease is generated on GAS that participates via several mechanisms to protect GAS against host innate defenses. The overall goal of this proposal centers on a unique combination of structural and biological studies of the productive interactions that mediate the binding and activation of hPg on M-Prts of GAS cells in vitro, ex vivo, and in vivo. The specific studies proposed to address this overall goal are contained in two Specific Aims, viz., Specific Aim 1. (a) To use biophysical approaches, e.g., SPR, to study the in vitro binding properties of hPg and to specific M-Prts, viz., PAM, as well as individual domains of this protein to minimize the primary sequences needed to provide the high affinity binding of hPg to PAM; (b) to study atomic level binding and dynamics of the PAM/hPg domains by high-resolution NMR and X-ray crystallography; and (c) to correlate the binding data with the activation of hPg on GAS cells containing PAM and variants of this protein to assess the nature of the productive binding of hPg to specific M-Prt domains. Hypothesis: these approaches will allow identification of the essential structural features of PAM that facilitate the binding and activation of hPg on specific types of GAS cells. Specific Aim 2. To generate and employ partially humanized mouse Pg transgenic mice, as well as transgenic bacteria with deletions of PAM and/or SK, to examine the effects of conscription of the fibrinolytic system on the stages of virulence of PAM-containing GAS. Hypothesis: using the knowledge gained in Aim 1, we will be capable of rationally designing mice and bacteria with altered virulence and identify the stages in which hPm bound to GAS exhibits its greatest effects. PHS Impact: A deepened understanding of the manner in which GAS interacts with the host innate immune system will allow approaches to be developed to attenuate the severity of GAS infections in humans.